SUMMARY: Cytosolic free calcium concentration [Ca] of protoplasts from was measured using the permeant acetoxy ester (quin-2-AM) of the calcium-chelating fluorescent dye quin-2. Low uptake of the ester occurred at pH 5.8-7.0 and its subsequent hydrolysis was low. Uptake was promoted by an external pH of 5.0 and significant hydrolysis to quin-2 achieved by adjustment of the internal pH to 7.2, which was near the optimum of the carboxylic esterases responsible for the hydrolysis. Uptake of Ca was biphasic with the average cell calcium concentration of protoplasts increasing from an initial value of 2 μmol to 50 μmol (kg cell water), during attainment of the steady state after 30 min, at which time [Ca] was unchanged at 20 nM but increased to 182 nM at 2-6 h exposure to 2.5 mM-Ca. Broadly similar changes in [Ca] were found in protoplasts derived from mycelium samples exposed to Ca over the same period of time. The location of Ca was determined in subfractionated organelles and characterized using enzyme markers and electron microscopy. In 32 h mycelium preloaded with Ca for 6 h, Ca was located principally in the mitochondria with lower concentrations associated with the endoplasmic reticulum, Golgi, vacuoles and plasma membrane components. Calcium was not released by inositol 1,4,5-trisphosphate or the calcium ionophore A23187 from any subcellular fractions obtained from mycelium on Percoll gradients, nor from preparations of vacuoles or plasmalemma vesicles, except in the case of mitochondria where rapid release of the ion was achieved by the addition of 2-5 μM-A23187. The anti-calmodulin agent calmidazolium (R24571) greatly reduced sporulation when addition preceded that of Ca. Calcium-induced cultures showed massive novel protein phosphorylation 2 h after addition of the ion which was virtually eliminated by R24571, whilst 1 h and 4-6 h protein phosphorylations, which were also present to some degree in vegetative controls, were substantially reduced. Two-dimensional SDS-PAGE analysis of phosphoproteins confirmed that Ca-induced mycelium had enhanced capacity for calmodulin-mediated phosphorylation relative to corresponding vegetative cells and that complex differential changes in such phosphorylations occurred during Ca-induction of the sporulation process.


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